The aim of this research is to investigate the binding of cholesterol oxidase to the membrane. Cholesterol oxidase is produced by some gram negative bacteria which can use cholesterol as a carbon source. Some of these organisms are pathogenic, such as Rhodococcus equi and Mycobacterium tuberculosis. It has been proposed that two surface residues, M332 and W333, contribute to binding by inserting their side chains into the hydrophobic core of the membrane. In order to test this hypothesis the following mutant enzymes will be made; M332A, M332C, W333A, W333C and W333F. The Cys mutants will be labeled with the fluorescent reporter groups acrylodan and monobromobimane. The mutant enzymes will be assayed for their ability to bind to vesicles of various lipid compositions under various conditions of ionic strength and pH. The mutants will also be tested for activity on cholesterol in micelles and in vesicles. The information gained from these studies will aid in improving serum- cholesterol assays, understanding the role of membrane cholesterol in receptor function and uncovering the role of cholesterol oxidase as a potential virulence factor.